Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021 is online at: average * The Journal of Immunology , 12 of which you can access for free at: 2012; 188:3088-3098; Prepublished online 20 from submission to initial decision 4 weeks from acceptance to publication February 2012; doi: 10.4049/jimmunol.1101764 http://www.jimmunol.org/content/188/7/3088 MF59 and Pam3CSK4 Boost Adaptive Responses to Influenza Subunit Vaccine through an IFN Type I-Independent Mechanism of Action Elena Caproni, Elaine Tritto, Mario Cortese, Alessandro Muzzi, Flaviana Mosca, Elisabetta Monaci, Barbara Baudner, Anja Seubert and Ennio De Gregorio J Immunol cites 33 articles Submit online. Every submission reviewed by practicing scientists ? is published twice each month by Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts http://jimmunol.org/subscription http://www.jimmunol.org/content/suppl/2012/02/21/jimmunol.110176 4.DC1 This article http://www.jimmunol.org/content/188/7/3088.full#ref-list-1 Information about subscribing to The JI No Triage! Fast Publication! Rapid Reviews! 30 days* Why • • • Material References Permissions Email Alerts Subscription Supplementary The Journal of Immunology The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2012 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. This information is current as of September 25, 2021. The Journal of Immunology MF59 and Pam3CSK4 Boost Adaptive Responses to Influenza Subunit Vaccine through an IFN Type I-Independent Mechanism of Action Elena Caproni,*,1 Elaine Tritto,†,1 Mario Cortese,* Alessandro Muzzi,* Flaviana Mosca,* Elisabetta Monaci,* Barbara Baudner,* Anja Seubert,* and Ennio De Gregorio* The innate immune pathways induced by adjuvants required to increase adaptive responses to influenza subunit vaccines are not well characterized. We profiled different TLR-independent (MF59 and alum) and TLR-dependent (CpG, resiquimod, and Pam3CSK4) adjuvants for the ability to increase the immunogenicity to a trivalent influenza seasonal subunit vaccine and to tetanus toxoid (TT) in mouse. Although all adjuvants boosted the Ab responses to TT, only MF59 and Pam3CSK4 were able to enhance hemagglutinin Ab responses. To identify innate immune correlates of adjuvanticity to influenza subunit vaccine, we investigated the gene signatures Downloaded from induced by each adjuvant in vitro in splenocytes and in vivo in muscle and lymph nodes using DNA microarrays. We found that flu adjuvanticity correlates with the upregulation of proinflammatory genes and other genes involved in leukocyte transendothelial migration at the vaccine injection site. Confocal and FACS analysis confirmed that MF59 and Pam3CSK4 were the strongest inducers of blood cell recruitment in the muscle compared with the other adjuvants tested. Even though it has been proposed that IFN type I is required for adjuvanticity to influenza vaccines, we found that MF59 and Pam3CSK4 were not good inducers of IFN-related innate immunity pathways. By contrast, resiquimod failed to enhance the adaptive response to flu despite a strong activation of the IFN http://www.jimmunol.org/ pathway in muscle and lymph nodes. By blocking IFN type I receptor through a mAb, we confirmed that the adjuvanticity of MF59 and Pam3CSK4 to a trivalent influenza vaccine and to TT is IFN independent. The Journal of Immunology, 2012, 188: 3088–3098. here are different types of influenza virus vaccines with protection against virus challenge (7). Alternatively, subunit flu distinct immunogenicity profiles (1–3). Live-attenuated vaccine formulation immunogenicity could be restored by incor- T virus vaccines induce a subclinical infection and there- poration of IFN inducers such as the TLR9 agonist (5). fore are very efficacious (4). Formalin-inactivated whole-virus Clinical experience has shown that the oil-in-water emulsions are vaccines (WV) contain viral RNA, which activates IFN type I extremely efficient in increasing the immunogenicity of flu subunit responses that are required to boost the adaptive immune re- vaccines. The adjuvant MF59 is an oil-in-water emulsion composed by guest on September 25, 2021 sponses (5). Subunit virus vaccines contain hemagglutinin (HA) of small droplets of squalene surrounded by a monolayer of nonionic and neuraminidase Ags, next to some residual structural proteins detergents (8, 9). Different aspects contribute to MF59 adjuvanticity. (M1 and NP), but lack most of the viral RNA, except from some Several studies have shown that MF59 can increase Ag uptake by residual RNA that may be associated with NP (6). These vaccines APCs (10–12) besides inducing the activation of innate immunity are safer compared with WV, but they are also less immunogenic (13). In vitro studies have shown that MF59 induces the secretion of and therefore require an adjuvant when administered to previously chemokines in human macrophages, monocytes, and granulocytes unexposed or immunocompromised subjects. It has been proposed (14). We have previously demonstrated that i.m. injection of MF59 that the best strategy to boost subunit flu vaccine formulation is to induces the local activation of innate immune reactions leading to restore IFN type I-associated activities that are lost during the recruitment of blood cells in the muscle (13). Recently, it has been purification process. Accordingly, coadministration of type I rIFN shown that all the recruited cell types can take up both Ag and with a subunit influenza vaccine in mice was shown to increase adjuvant and that MF59 promotes the migration of Ag/adjuvant double-positive cells to the draining lymph nodes (LNs) (15). A MF59-adjuvanted influenza vaccine was developed initially *Research Center, Novartis Vaccine and Diagnostics, 53100 Siena, Italy; and for seasonal vaccination in the elderly to enhance Ab titers and †Preclinical Safety Genomics, Novartis Institute of Biomedical Research, CH-4057 seroconversion and seroprotection rates (9). More recently it was Basel, Switzerland shown that also in young children MF59 enhances seroconver- 1 E.C. and E.T. contributed equally to this study. sion and cross-protection against flu strains mismatched to the Received for publication June 17, 2011. Accepted for publication January 13, 2012. Ags of a seasonal subunit flu vaccine (16). MF59 and a second The microarray data presented in this article have been submitted to the ArrayExpress oil-in-water emulsion, called AS03, have also been successfully database, European Molecular Biology Laboratory-European Bioinformatics (http:// www.ebi.ac.uk/), under accession number E-MTAB-942. used for the development of prepandemic flu vaccines to protect from a potential H5N1 pandemic influenza outbreak (17, 18). The Address correspondence and reprint requests to Dr. Ennio De Gregorio, Novartis Vaccine and Diagnostics, Via Fiorentina 1, 53100 Siena, Italy. E-mail address: clinical data have shown that MF59 increases cross-reactive neu- [email protected] tralizing Ab titers and memory responses to H5 (17, 19). Recently, The online version of this article contains supplemental material. MF59 and AS03 have also been used during the vaccination cam- Abbreviations used in this article: DC, dendritic cell; HA, hemagglutinin; HI, hem- paign in the 2009–2010 H1N1 pandemic outbreak (20). agglutination inhibition; KO, knockout; LN, lymph node; TT, tetanus toxoid; WV, The ability of emulsions to increase the immunogenicity of sub- whole-virus vaccine. unit flu vaccines seems to be quite unique. Clinical trials have shown Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00 that the administration of MF59-adjuvanted H5N1 induces higher www.jimmunol.org/cgi/doi/10.4049/jimmunol.1101764 The Journal of Immunology 3089 hemagglutination inhibition (HI) and microneutralization titers to measured by endpoint ELISA. MaxiSorp 96-well plates were coated with flu compared with the administration of the same vaccine formulated 2.5 mg/well flu Ag or 0.2 mg/well TT Ag in 100 ml/well PBS. Goat anti- with alum (21). Furthermore, mouse studies have shown that MF59 mouse IgG (Southern Biotechnology Associates), goat anti-mouse IgG1 (Southern Biotech), or goat anti-mouse IgG2a (Southern Biotechnology enhances HI titers to seasonal flu Ags compared with alum and CpG Associates) Abs, all conjugated with alkaline phosphatase, were used at (22). However, the innate immune pathways required for the adju- 1:2000 dilution. The reaction was developed by addition of 100 ml/well vant effect of oil-in-water emulsions to influenza subunit vaccines p-nitrophenyl phosphate disodium salt substrate (Sigma-Aldrich), and ab- and the exact role played by IFN type I are not well characterized. sorbance at 405 nm was measured by Spectramax plate reader. ELISA titers were expressed as the reciprocal dilution that gave an OD higher than In this work, we investigated the innate immune reactions that are the average OD of the blanks plus 3 times the SD. associated with adjuvanticity to influenza subunit vaccines in mouse using a systems biology approach. First, we monitored the ability of Measurement of flu HI inhibition titers MF59, alum, and three TLR-dependent adjuvants, CpG, resiquimod, Sera were pretreated with DENKA receptor-destroying enzyme (Bio- and Pam3CSK4, to increase